An improved design of the kissing complex-based aptasensor for the detection of adenosine

Abstract

We very recently reported a novel aptamer biosensing concept based on a dual recognition mechanism originating from the small target-induced formation of a functional nucleic acid assembly. This assembly is constituted of a hairpin aptamer (named aptaswitch) for which the apical loop of the parent aptamer is substituted by a short RNA sequence prone to loop-loop interactions. It can switch between folded and unfolded states in the presence and in the absence of targets, respectively. The apical loop of the folded aptaswitch is then recognized by a second hairpin (called aptakiss), forming a kissing complex that signals the presence of the target. In the present work, we focus on the design improvement of this biosensing platform by using a previously described adenosine-adenoswitch couple as a model system and a fluorophore-labeled aptakiss as a reporting probe for fluorescence anisotropy (FA) detection. In the first step, the initially described adenoswitch was re-engineered to optimally convert the unfolded structure into the active stem-loop form upon adenosine binding. To further improve the assay performance, a blocking DNA oligonucleotide of the adenoswitch sequence was subsequently introduced into the assay scheme. This blocking strategy led to a significant increase in the FA response by reducing the background signal generated by the undesired binding of the free adenoswitch to the aptakiss probe. We obtained a detection limit which is fivefold lower than that observed with the previously reported kissing complex-based sensor. Finally, the optimized biosensing platform was successfully applied under biologically relevant conditions, i.e., diluted human serum, suggesting the potential practical applicability of the kissing sensing approach.

ELAKCA: Enzyme-Linked Aptamer Kissing Complex Assay as a Small Molecule Sensing Platform

“…In the first stage of the work, we aimed to demonstrate the proof-of-principle of ELAKCA by using the aptaswitch-aptakiss couple specific to adenosine ( ADOsw-28 / aptakiss-k1 , see Table S1 for sequences). These functional oligonucleotides have been previously constructed to develop other kinds of kissing complex-based aptamer sensing platforms. , Their design involved different key steps. The kissing motifs were initially identified from an artificial repertoire of RNA hairpins .…”

“…The aptamer is engineered in such a way that the binding of the small target shifts its conformation from an unfolded to a folded hairpin shape (aptaswitch). The apical loop of the parent aptamer is substituted by a short RNA sequence (in blue in Figure ) liable to interact with the loop of a second RNA hairpin (aptakiss) so that the folded structure of the aptaswitch is recognized through the formation of a kissing complex. − The aptaswitch is then immobilized onto the well surface to serve as the target capture element whereas the aptakiss is labeled to allow the enzyme-linked target detection by establishing the specific loop–loop interaction with the target-bound aptaswitch.…”

Single-molecule observations of RNA–RNA kissing interactions in a DNA nanostructure

“…[1][2][3][4][5][6][7][8] This specific kissing complex formation with native RNA motifs has been used to create various RNA architectures such as polygonal structures and three-dimensional assemblies. One of the variations of the complex formation between RNA molecules includes a "kissing complex," which enables assembly of complementary RNA loops via Watson-Crick base pairing.…”

“…One of the variations of the complex formation between RNA molecules includes a "kissing complex," which enables assembly of complementary RNA loops via Watson-Crick base pairing. [1][2][3][4][5][6][7][8] This specific kissing complex formation with native RNA motifs has been used to create various RNA architectures such as polygonal structures and three-dimensional assemblies. [9][10][11] Aptamers with a kissing RNA loop have been artificially developed by in vitro selection.…”

A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers

“…Different designs were described that involve a monomolecular ( 45 ), a bimolecular ( 46 ) or even a trimolecular architecture ( 47 ) that ultimately translates binding into a signal, fluorescence energy transfer for instance. We recently described a new type of aptasensor relying on loop–loop (kissing) interactions ( 48 , 49 ): the binding of the ligand to an imperfect RNA hairpin aptamer named aptaswitch specifically triggers the formation of a kissing complex with a second RNA stem-loop termed aptakiss. This concept can be extended to the simultaneous detection of several molecules as far as specific aptaswitches are available for each of them on the one hand and several kissing sequences are identified on the other hand.…”